This invention relates to an apparatus and method for producing microarrays of biological materials.
Microarrays of biological materials comprise a support, such as chemically activated paper or glass, on which is arranged a number of small discrete deposits of biological materials such as DNA, RNA, or proteins, in predetermined patterns or arrays. Microarrays can be used for a number of different purposes, including the identification of unknown samples of materials such as DNA and antibodies.
There are two major approaches to the production of microarrays: synthesis and delivery. The synthesis approach involves the production of microarrays in a step-wise fashion by the in situ synthesis of nucleic acids and other biopolymers from biochemical building blocks, such that with each round of synthesis, building blocks are added to growing chains until the desired length is achieved. The delivery approaches, by contrast, use the exogenous deposition of pre-prepared biochemical substances for chip fabrication.
At least three types of technologies are known in the art for automated microarray production. These are photolithography, mechanical microspotting, and ink jet technology. With photolithography, a glass wafer, modified with photolabile protecting groups is selectively activated for DNA synthesis by shining light through a photomask. Repeated deprotection and coupling cycles enable the preparation of high-density oligonucleotide microarrays (see for example, U.S. Pat. No. 5,744,305, issued Apr. 28, 1998).
Microspotting encompasses deposition technologies that enable automated microarray production by printing small quantities of pre-made biochemical substances onto solid surfaces. Printing is accomplished by direct surface contact between the printing substrate and a delivery mechanism, such as a pin or a capillary. Robotic control systems and multiplexed printheads allow automated microarray fabrication.
Ink jet technologies utilize piezoelectric and other forms of propulsion to transfer biochemical substances from miniature nozzles to solid surfaces. Using piezoelectricity, the sample is expelled by passing an electric current through a piezoelectric crystal which expands to expel the sample. Piezoelectric propulsion technologies include continuous and drop-on-demand devices. In addition to piezoelectric ink jets, heat may be used to form and propel drops of fluid using bubble-jet or thermal ink jet heads; however, such thermal ink jets are typically not suitable for the transfer of biological materials due to the heat which is often stressful on biological samples. Examples of the use of ink jet technology include U.S. Pat. No. 5,658,802 (issued Aug. 19, 1997).
The apparatus presently available often suffers from one or more of the following disadvantages: high cost, lack of automatization, sample cross-contamination the requirement for large volumes of sample, and lack of versatility, for instance in the amount and shape of the sample deposited on the microarray. There is, therefore, a need for new methods and apparatus for preparing microarrays. Ideally, such methods should utilize relatively simple and inexpensive machinery to produce the microarrays in a reproducible and rapid manner.
In one aspect, the invention provides an apparatus for forming a microarray of biological materials on a substrate, comprising: a support adapted to hold the substrate; at least one electro-mechanical fluid ejector, adapted to deliver a sample to the substrate held in said support; a plurality of sample reservoirs; one or more sampling devices, adapted to take up a sample from a sample reservoir; a positioning device for moving the plurality of sample reservoirs and the sampling device relative to each other, so as to position the sampling device adjacent to a selected sample for take up of the selected sample by the sampling device; a conduit for delivering the selected sample taken up in the sampling device to said ejector; and a transport mechanism for moving the substrate-holding support and the ejector relative to each other, such that the mircoarray is produced by delivery of samples to the substrate in a predetermined pattern.
Advantages of the subject invention include the complete automatization of the process, such that samples do not have to be replaced or exchanged during the process. Each sampling device is able to pick up sample from any one of the sample reservoirs and deliver it to an ejector. Thus, a large assortment of samples can be dispensed to form a microarray. Further, computer control can be used, so that no human intervention is required during a run.
Electro-mechanical fluid ejectors (ink jet) are used to deposit small drops of liquid on a solid substrate at rates much more rapid than those achievable with microspotting devices. In addition, such ejectors allow the use of extremely small samples (10 pl to 1 nl). Thus, with an accurate positioning mechanism, microarrays can be formed which have a larger number of probes located within a smaller area than is achievable with many prior methods. The use of extremely small drops also minimizes costs by minimizing the quantity of sample required.
The apparatus itself can be assembled from commercially available components which also make it much more inexpensive than prior art methods which require expensive components manufactured specifically for making microarrays.
In preferred aspects, the invention provides for cleaning of the conduit line through which line the sample is transferred from the sampling device to the ejector, as well as for cleaning and drying of the exterior of the sampling device and the ejector. This provides a means of avoiding contamination, such that a sampling device can be used to pick up and successively deliver any number of different samples to the ejector. This allows for the use of many different samples, again without human intervention being necessary during a run. This also increases the speed at which a microarray can be produced.
In another aspect, the invention provides a commercial package comprising components of the apparatus as described above, together with instructions for the assembly and use of the apparatus.
In a further aspect, the invention provides a method for forming a microarray of biological materials on a substrate, comprising: providing a plurality of sample reservoirs which contain samples; providing a sampling device; positioning the sampling device adjacent to the sample reservoirs; using the sampling device to draw a selected sample into the sampling device from the sample reservoirs; delivering said selected sample to an ejector through a conduit; providing a substrate positioning the ejector adjacent to said substrate; delivering the sample from the ejector to a predetermined location on the substrate; repeating the above steps such that multiple samples are delivered to the substrate at pre-determined locations to form a microarray of biological materials.